Biliverdin from a Non-Animal Source

ABSTRACT

Methods for producing biliverdin in a microorganism, methods for producing biliverdin from a non-animal source, cells for producing biliverdin and methods for producing cells for producing biliverdin are disclosed.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 61/258,126, filed Nov. 4, 2009, entitled “Enhanced Bioproduction of Biliverdin-IX alpha,” which is hereby incorporated herein by reference.

BACKGROUND

Biliverdin IX-alpha (biliverdin IXα) is the most common form of several biliverdin isomers found in nature (FIG. 1). It is produced from heme (also named iron protoporphyrin IX) (FIG. 1) by the enzyme heme oxygenase. In animals, biliverdin IXα is reduced by the enzyme biliverdin reductase to bilirubin IXα which is the major form of several bilirubin isomers (FIG. 1). One known role of biliverdin IXα in nature is in animals as an intermediate in hemoglobin breakdown as red blood cells are degraded in phagocytes (FIG. 2). The hemoglobin prosthetic group, heme, with its bound iron, is released in this degradative process, and heme is converted by HO to biliverdin IXα. Biliverdin IXα is then converted to bilirubin IXα by the enzyme biliverdin reductase (FIG. 2). Bilirubin IXα is consecutively bound to serum albumin and then in the liver to glucoronic acid (conjugated bilirubin) which confers a relatively high degree of water solubility. Conjugated bilirubin is then excreted in the bile. Overall, this process is viewed as a process for animals (e.g. humans) to degrade and eliminate heme—which is toxic when accumulated.

Biliverdin IXα is also made by microbes. For example, biliverdin IXα is a precursor to microbial phycobilins, i.e. phycocyanobilin (pcb) and phycoerythrobilin (peb). Pcb and peb are the pigment molecules for the light-harvesting complexes of photosynthetic cyanobacteria, phycocyanin and phycoerythrin, respectively. These complexes collect light energy (for example solar energy) and funnel it to photosynthetic reaction centers where the energy is converted into chemical energy) (FIG. 3). Pcb is also the pigment for phytochrome—a light sensing receptor that occurs in plants and other cells. An analogous receptor, bacteriophytochrome, is found in certain bacteria. The pigment for bacteriophytochrome is biliverdin IXα rather than pcb, which reveals yet another biological role for biliverdin IXα. These latter bacteria—like all microbes—either do not produce or do not accumulate bilirubin IXα. The lack of bilirubin IXα accumulation by microbes is either a consequence of lacking biliverdin reductase or the conversion of bilirubin IXα to bile pigments such as those involved in photosynthetic light-harvesting.

Bilirubin IXα is known to associate with cell membranes where it quenches the propagation of reactive oxygen species (ROS). It is therefore believed to confer protection to membrane lipid and protein components against oxidative damage. Thus, an additional suggested function of biliverdin IXα is to serve as the immediate source of bilirubin IXα which in turn acts as a cytoprotective antioxidant and anti-inflammatory agent against cell damaging ROS (FIG. 4). Although bilirubin IXα (and not biliverdin IXα) is believed to be the cytoprotective antioxidant, it is observed that biliverdin IXα is more effective than bilirubin IXα when administered at tissue injury/inflammatory sites where ROS are prevalent. One explanation for the higher efficacy of biliverdin IXα is that it is more hydrophilic than bilirubin IXα and therefore has better access to tissue sites where it is then reduced by biliverdin reductase to bilirubin IXα. Another explanation is that when biliverdin IXα binds to biliverdin reductase, this enzyme is activated and initiates a cell signaling cascade that results in the production of the anti-inflammatory cytokine interferon-10.

There is increasing evidence that biliverdin IXα can be used as a cytoprotective therapeutic agent. Examples of clinical applications of biliverdin IXα include treatment of vasoconstriction (U.S. Patent Application Publication No. 20030027124); coronary artery disease (artherosclerosis); ischemia/reperfusion injuries after small intestinal, heart, and kidney transplantation; severe sepsis; injuries from liver grafts; and prevention of intimal hyperplasia induced by vascular injury. Today, biliverdin IXα is predominantly derived by chemical oxidation of bilirubin IXα or by using the enzyme bilirubin oxidase (U.S. Pat. No. 5,624,811). Bilirubin IXα is extracted from the bile of various mammals, especially from swine or other livestock. Commercial animal bilirubin IXα preparations are often contaminated with conjugated bilirubin and isomers (e.g. bilirubin XIIIα) (Reisinger et al. 2001; U.S. Pat. No. 431,166). As a result, biliverdin IXα derived from bilirubin IXα preparations using oxidative processes or enzymes may also contain isomers. The clinical consequences of using biliverdin IXα contaminated with such isomers are not clear. In addition, the use of biliverdin IXα preparations derived from animal bilirubin carries the risk of prion contamination often associated with materials derived from animal sources.

A recent claim (U.S. Pat. No. 7,504,243) for biliverdin IXα production by a yeast depends on addition of hemoglobin (from animal blood) to the growth culture as a source of heme. Another report shows biliverdin IXα synthesis by Escherichia coli expressing a heterologous HO gene of animal origin (rat). The biliverdin IXα was produced at low levels and appears to remain cell-bound.

The limited amounts of biliverdin IXα produced by yeast and E. coli expressing heterologous HO genes could result from restricted access to heme. In E. coli, the biosynthesis of heme is regulated at the initial step of tetrapyrrole biosynthesis—the synthesis of 5-aminolevulinic acid (ALA) by the C5-pathway. The C5 pathway involves conversion of glutamate to glutamyl-tRNA by glutamyl-tRNA synthetase, reduction to glutamate-γ-semialdehyde by an NADPH-dependent glutamyl tRNA reducatase and transamination by glutamate-γ-semialdehyde aminomutase to form ALA. The C5 pathway is feedback-inhibited by heme and, as a consequence, the cellular levels of heme are kept low. In contrast, mammals, plants, and certain bacteria such as Rhodobacter sphaeroides produce ALA from glycine and succinyl-CoA via the enzyme ALA synthetase. This latter mechanism for ALA biosynthesis is termed the “C4 pathway.” The C4 pathway ALA synthetase is not subject to feedback inhibition by heme. It therefore allows the accumulation of heme and higher cellular concentrations. When combined with the C4 pathway for ALA synthesis, HO will have greater access to its substrate, heme, resulting in increased potential for producing biliverdin IXα.

SUMMARY

Methods for producing biliverdin in a microorganism, methods for producing biliverdin from a non-animal source, cells for producing biliverdin and methods for producing cells for producing biliverdin are disclosed. These methods and cells conform to a general strategy for enhanced production of biliverdin for a non animal source. This general strategy is depicted in FIG. 14.

In one aspect, a method of producing biliverdin in a microorganism is disclosed, comprising: culturing a cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.

In certain embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase. In certain other embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme. The cell may also comprise a regulatable promoter operably linked to a polynucleotide which codes for the heme oxygenase. In a related embodiment, the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme biosynthetic enzyme. The regulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase. In a related embodiment, the heme biosynthetic enzyme is hemA. In another related embodiment, the heme biosynthetic enzyme is an ALA synthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme. In a related embodiment, the heme oxygenase enzyme is HO1. In another related embodiment, the heme oxygenase enzyme is an HO family analog.

In certain other embodiments, lactose is provided to the cell. Lactose may be provided in an initial concentration of from about 2% (w/v) to about 10% (w/v). Furthermore, the methods may further comprise the step of providing a trace metal to the cell. Trace metals may be added as part of a trace metal solution. Some or all of NaCl, ZnSO₄, MnCl₂, FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine or CaCl₂ may be provided to the cell.

In certain other embodiments, a plurality of measurements of the dissolved oxygen concentration in the growth medium of the cell may be taken. In a related set of embodiments, at least an initial measurement, a first intermediate measurement, a second intermediate measurement, and a third intermediate measurement of dissolved oxygen concentration in the growth medium of the cell may be taken. In related embodiments, the initial measurement yields an initial value, the first intermediate measurement yields a first intermediate value less than the initial value, the second intermediate measurement yields a second intermediate value greater than the first intermediate value, and the third intermediate measurement yields a third intermediate value less than the first intermediate value. In further related embodiments, dissolved oxygen in the growth medium in concentrations at the initial value, the first intermediate value, the second intermediate value and the third intermediate value indicates that biliverdin will be produced.

In certain other embodiments, the biliverdin is excreted into the growth medium of the cell. In one such embodiment, foam accumulates in the growth medium of the cell and a portion of the biliverdin excreted in to the growth medium of the cell is located with the foam. In related embodiments, the foam is collected. In any embodiment, the biliverdin may be collected.

In another aspect, a method of producing biliverdin from a non-animal source is disclosed, comprising: culturing a cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.

In certain embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase. In certain other embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme. The cell may also comprise a regulatable promoter operably linked to a polynucleotide which codes for the heme oxygenase. In a related embodiment, the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme biosynthetic enzyme. The regulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase. In a related embodiment, the heme biosynthetic enzyme is hemA. In another related embodiment, the heme biosynthetic enzyme is an ALA synthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme. In a related embodiment, the heme oxygenase enzyme is HO1. In another related embodiment, the heme oxygenase enzyme is an HO family analog.

In certain other embodiments, lactose is provided to the cell. Lactose may be provided in an initial concentration of from about 2% (w/v) to about 10% (w/v). Furthermore, the methods may further comprise the step of providing a trace metal to the cell. Trace metals may be added as part of a trace metal solution. Some or all of NaCl, ZnSO₄, MnCl₂, FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine or CaCl2 amy be provided to the cell.

In certain other embodiments, a plurality of measurements of the dissolved oxygen concentration in the growth medium of the cell may be taken. In a related set of embodiments, at least an initial measurement, a first intermediate measurement, a second intermediate measurement, and a third intermediate measurement of dissolved oxygen concentration in the growth medium of the cell may be taken. In related embodiments, the initial measurement yields an initial value, the first intermediate measurement yields a first intermediate value less than the initial value, the second intermediate measurement yields a second intermediate value greater than the first intermediate value, and the third intermediate measurement yields a third intermediate value less than the first intermediate value. In further related embodiments, dissolved oxygen in the growth medium in concentrations at the initial value, the first intermediate value, the second intermediate value and the third intermediate value indicates that biliverdin will be produced.

In certain other embodiments, the biliverdin is excreted into the growth medium of the cell. In one such embodiment, foam accumulates in the growth medium of the cell and a portion of the biliverdin excreted in to the growth medium of the cell is located with the foam. In related embodiments, the foam is collected. In any embodiment, the biliverdin may be collected.

In another aspect, a cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme is disclosed.

In certain embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase. In certain other embodiments, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme. The cell may also comprise a regulatable promoter operably linked to a polynucleotide which codes for the heme oxygenase. In a related embodiment, the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme biosynthetic enzyme. The regulatable promoter may comprise the T7 promoter.

In certain embodiments, the heme biosynthetic enzyme is an ALA synthase. In a related embodiment, the heme biosynthetic enzyme is hemA. In another related embodiment, the heme biosynthetic enzyme is an ALA synthase analog.

In certain other embodiments, the heme oxygenase is a HO family enzyme. In a related embodiment, the heme oxygenase enzyme is HO1. In another related embodiment, the heme oxygenase enzyme is an HO family analog.

In another aspect, methods of producing a cell for producing biliverdin are disclosed. In certain embodiments, a polynucleotide comprising a sequence which codes for a recombinant heme oxygenase is introduced into a parent cell comprising a recombinant heme biosynthetic enzyme. In certain other embodiments, a polynucleotide comprising a sequence which codes for a recombinant heme biosynthetic enzyme is introduced into a parent cell comprising a recombinant heme oxygenase. In certain other embodiments, the method comprises introducing into a parent cell a first polynucleotide comprising a first sequence which codes for a recombinant heme oxygenase; and a second polynucleotide comprising a second sequence which codes for a recombinant heme biosynthetic enzyme.

In other embodiments, a polynucleotide comprising a first sequence which codes for a recombinant heme oxygenase and a second sequence which codes for a recombinant heme biosynthetic enzyme is introduced into a parent cell.

In certain other embodiments, a first polynucleotide comprising a promoter sequence is introduced into a parent cell, and the first polynucleotide recombines with a second polynucleotide comprising a sequence which codes for a heme oxygenase, such that the first polynucleotide and a portion of the second polynucleotide form a linked polynucleotide which codes for a recombinant heme oxygenase.

In another set of embodiments, a first polynucleotide comprising a promoter sequence is introduced into a parent cell, and the first polynucleotide recombines with a second polynucleotide comprising a sequence which codes for a heme biosynthetic enzyme, such that the first polynucleotide and a portion of the second polynucleotide form a linked polynucleotide which codes for a recombinant heme biosynthetic enzyme.

For any method of producing a cell for producing biliverdin, introducing a polynulcoetide into a cell may be performed by transformation. The cell used may be a microorganism, bacterial cell, or an Escherichia coli cell. Furthermore, the methods of producing biliverdin may comprise culturing cells comprising a polynucleotide which has been optimized for expression in a cell. The cells for producing biliverdin may comprise culturing cells comprising a polynucleotide which has been optimized for expression in a cell. Furthermore, the methods of producing cells for producing biliverdin may comprise introducing a polynucleotide into the parent cell, the polynucleotide having been optimized for expression in a cell.

Additional aspects and advantages will be apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of heme (top), bilirubin IXα (center), and biliverdin IXα (bottom).

FIG. 2 shows the metabolic transformations of heme, biliverdin IXα and bilirubin IXα in animals.

FIG. 3 shows the roles of biliverdin IXα and HO-1 in the biosynthesis of cyanobacterial phycocyanobilins.

FIG. 4 shows the cytoprotective role of biliverdin IXα and bilirubin IXα against ROS. Acronym definitions are: HO (heme oxygenase), BVR (biliverdin reductase) and ROS (reactive oxygen species). The bars indicate suppression of ROS.

FIG. 5 shows a gene map for pET101 plasmid expression vector with T7 promoter used to express HemA and HO1 in E. coli.

FIG. 6 shows the DNA base sequence of cyanobacterium Synechocystis PCC6803 HO1 (SEQ ID NO: 48) (726 base pairs).

FIG. 7 shows the DNA base sequence of HemA of R. sphaeroides ALA synthetase (SEQ ID NO: 49) (1224 base pairs).

FIG. 8 shows the chemical structure of isopropyl-beta-D-thiogalactopyranoside (IPTG).

FIG. 9 shows a gene map of the plasmid expression vector HemA-HO1-pET101 with HO1 and HemA genes inserted downstream of DNA sequences that permit transcriptional regulation via T7 lac operon dependent mechanisms and ribosome binding sites (RBSs) for each gene to provide efficient initiation of protein translation.

FIG. 10 shows a photograph of solid phase extraction Sepak C18 column (3 ml) with green material produced by E. coli (HO-1). A methanol extract of green aggregated material from growing cultures was loaded in 40% methanol, 0.2M Na acetate, pH 5.2. The green material was then eluted off the column with 100% methanol and recovered.

FIG. 11 shows absorbance spectra of a) methanol-extracted green material produced by E. coli (HO1), loaded onto a C18 solid phase extraction Sepak C-18 column (3 ml) in 40% methanol, 0.2 M Na acetate, pH 5.2, and recovered by elution with 100% methanol, and b) biliverdin IXα standard, commercially available from Frontier Scientific, Inc.

FIG. 12A shows a high-performance liquid chromatograms of methanol-extracted green material from cultures of E. coli (HO1)

FIG. 12B shows a high-performance liquid chromatograms of a biliverdin IXα standard, commercially available from Frontier Scientific, Inc. The bacterially-derived green material (FIG. 12A) contains components with retention times that are identical to commercially available biliverdin IXα.

FIG. 13A shows a mass spectrum showing the mass of the methanol, extracted and solid phase extraction recovered “green material” from E. coli (HO1).

FIG. 13B shows a mass spectrum showing the mass of a commercial biliverdin IXα standard obtained from Frontier Scientific, Inc. The methanol extracted and solid phase extraction recovered “green material” from E. coli (HO1) (FIG. 13A) has the same mass (m/e 583.68) as a commercial biliverdin IXα standard obtained from Frontier Scientific, Inc. (FIG. 13B).

FIG. 14 shows an overall strategy of increasing heme oxygenase activity and the biosynthesis of heme in a host cell to enhance Biliverdin IX alpha accumulation.

FIG. 15 shows dissolved oxygen (DO-1) and pH (pH-1) in growth medium of Escherichia coli cells during biliverdin production.

FIG. 16A through FIG. 16B shows an alignment of several HO family heme oxygenases.

FIG. 17 shows an alignment of several subfamily members for several HO subfamilies. FIG. 17A shows an alignment of the HO-1 subfamily of heme oxygenases. FIG. 17B through FIG. 17C shows an alignment of the HMOX1 subfamily of heme oxygenases. FIG. 17D shows an alignment of the ho subfamily of heme oxygenases.

FIG. 18A through FIG. 18C shows an alignment of diverse ALA synthases.

FIG. 19A through FIG. 19C shows an alignment of ALA synthases that are homologs of SEQ ID NO: 2.

FIG. 20 shows the optimized HO-1 gene DNA sequence provided by DNA 2.0 Inc. (SEQ ID NO: 50).

FIG. 21 shows the polypeptide sequence of HO-1 (SEQ ID NO: 1)

FIG. 22 shows a gene map for the vector HO1-pJ401.

FIG. 23 shows the polypeptide sequence of HemA (SEQ ID NO: 2).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

“Biliverdin” means biliverdin IXα (CAS Registry Number: 114-25-0).

To “culture” or “culturing” means to provide nutrients to a cell sufficient to allow the cell to grow and reproduce. Methods of culturing cells are known in the art. In particular, method of culturing cells, including bacteria, are described in (Joe Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, 2001 (ISBN: 0879695773); or in Frederick M. Ausubel, et al., eds., Current Protocols in Molecular Biology, Wiley (ISSN: 1934-3639, last updated Jun. 28, 2010)), which are incorporated by reference.

“Recombinant” may be used to describe a polynucleotide or a polypeptide. A “recombinant polynucleotide” is a polynucleotide within a cell that comprises a sequence not naturally found inside that cell. For example, a recombinant polynucleotide could comprise a coding sequence (CDS) that is not naturally found in the cell. Alternatively a recombinant polynucleotide could comprise two sequences joined together, where one sequence is naturally found in the cell, and the other is not. For example a CDS naturally found in the cell could be operably linked to a promoter that is not naturally found in the cell. A “recombinant polypeptide” is the polypeptide product of a recombinant polynucleotide that comprises a CDS. For example, a recombinant heme oxygenase is the polypeptide product of a recombinant polynucleotide which codes for the heme oxygenase.

A polynucleotide “codes for” a polypeptide when the polynucleotide comprises a set of codons which, when transcribed and translated by cellular machinery, will produce a polypeptide whose amino acid sequence corresponds to the codons of the polynucleotide according to a genetic code.

“CDS” means coding sequence. A coding sequence is a polynucleotide sequence that codes for a polypeptide product.

A polynucleotide or polypeptide is “naturally found” in a cell when that polynucleotide or polypeptide is present in a healthy, uninfected, wild-type cell under one or more culture conditions. All other polynucleotides and polypeptides are not naturally found in a cell.

A “heme oxygenase” is an enzyme with the activity defined by Enzyme Commission (“E.C.”) number 1.14.99.3. Heme oxygenases include two families of enzymes, the HO family, and the HemS family. The HO family is a defined enzyme family comprising, for example, polypeptides (SEQ ID NOs: 1 and 7-28). The identity of these SEQ IDs is shown in Table 1. The HO family may be defined, for example, by shared sequence motifs as described in the Hidden Markov Model Pf01126. An alignment of several HO family heme oxygenases is provided in FIG. 16. The HO family may also be subdivided into subfamilies, including the HMOX1 subfamily found in some animals, the HO-1 subfamily found in some cyanobacteria and the ho subfamily found in some plants. An alignment of several subfamily members for several HO subfamilies is found in FIG. 17. The HemS family of enzymes is also a defined enzyme family comprising Escherichia coli O157:H7 gene product ChuS and other proteins. The HemS family may be defined, for example, by shared sequence motifs as described in the Hidden Markov Model PF05171.

TABLE 1 Representative sample of HO family heme oxygenases SEQ ID Species Gene product 1 Synechocyctis PCC 6803 HO-1 7 Cyanotheca sp. cce2573 8 Synechococcus sp. A2508 9 Anabaena sp. all1897 10 Corynebacterium Diphtheriae Hmuo 11 Synechocystis sp. 1WOW_A 12 Oryctolagus cuniculus heme oxygenase 2 13 Pseudomonas aeruginosa gi 50513550 14 Takifugu rubripes HMOX 15 Drosophila melanogaster Q9VGJ9 16 Bradyrhizobium sp gi 75412672 17 Pseudomonas aeruginosa gi 81540044 18 Streptomyces coelicolor gi 81550417 19 Bos taurus HMOX1 20 Homo sapiens HMOX1 21 Mus musculus Hmox1 22 Rattus norvegicus Hmox1 23 Arabidopsis thaliana ho4 24 Arabidopsis thaliana ho3 25 Gallus gallus HMOX1 26 Danio rerio hmox1 27 Pan troglodytes HMOX1 28 Canis lupus familiaris HMOX1

A “heme oxygenase analog” means a heme oxygenase enzyme bearing one or more additions deletions or substitutions of residues compared to the original heme oxygenase. Useful heme oxygenase analogs include analogs which retain the heme oxygenase activity defined by E.C. 1.14.99.3. By examining and aligning known heme oxygenase sequences from a given heme oxygenase family, a skilled person can determine which heme oxygenase residues are conserved across species. Using this alignment, the skilled person could generate a consensus sequence, using, for example, the Clustal algorithm. Since conserved residues are generally those which are required for function (Boffelli D, Nobrega M A, Rubin E M. Comparative genomics at the vertebrate extremes. Nat Rev Genet. 2004; 5:456-465), non-naturally occurring proteins that conform to this consensus sequence would define heme oxygenase analogs that likely retain the heme oxygenase activity defined by E.C. 1.14.99.3. For example, from the alignments in FIG. 16 or 17, a consensus sequence for the HO family could be constructed. Further, additional alignments may be generated using, for example, the HomoloGene and Conserved Domain (CDD) algorithms of the National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda Md. USA.

Alternatively, generating heme oxygenase analogs which retain heme oxygenase activity could also be accomplished by using existing bioinformatic resources. Proteins and protein domains are often described by a Hidden Markov Model (HMM). An HMM of a polypeptide is not a sequence alignment, but it does convey actual structural information about the protein. Most HMMs are based on the probability of any particular residue occurring next to a second residue in the linear sequence of the polypeptide. Using HMMs to describe proteins is discussed in Krogh A, Brown M, Mian I S, Sjölander K, Haussler D, Hidden Markov models in computational biology. Applications to protein modeling. J Mol. Biol. 1994; 235; 1501-31, which is hereby incorporated by reference.

The European Bioinformatics Institute maintains the Interpro database, which compiles HMM information from various databases, including some described below. Interpro has two different entries which describe heme oxygenases. The first is IPR002051 Haem oxygenase (defining the HO family) and IPR007845 Haemin-degrading HemS (defining the HemS family).

The Wellcome Trust Sanger Institute also maintains the Pfam database, which describes the heme oxygenase proteins in terms of HMMs. The Pfam HMMs that define the heme oxygenase proteins are PF01126 (HO family) and PF05171(HemS family). Included in the database for each HMM entry is a feature which allows the user to visualize the structural information in the HMM.

Although the HMMs do not provide typical sequence information regarding heme oxygenase proteins, they do provide a description of the probable structure of a heme oxygenase. Thus, analogs of heme oxygenase that conform to the HMM would be more likely to retain heme oxygenase activity. To easily generate sequences of heme oxygenase analogs more likely to have heme oxygenase activity, a skilled person could generate heme oxygenase analog sequences using a computer to introduce substitutions, deletions or additions to a heme oxygenase sequence. The relative probabilities embodied in the heme oxygenase HMMs could guide a skilled person regarding which residues, when mutated, are more likely to lead to a loss of function. The skilled person could then compare the analog sequences to the HMMs in the databases listed above. Those analogs which met the threshold of being tagged as bearing a heme oxygenase domain would likely have the property of heme oxygenase activity. The HMMs discussed above which describe heme oxygenases and heme oxygenase analogs are hereby incorporated by reference.

“HO1” or “HO-1” is the polypeptide represented by SEQ ID NO: 1.

A “heme biosynthetic enzyme” is an enzyme involved in the anabolic metabolism of heme. Heme biosynthetic enzymes include Amino levulinic acid dehydratase, Porphobilinogen deaminase, Uroporphyrinogen III synthase, Uroporphyrinogen III decarboxylase, Coprophorinogen III oxidase, Protopophyrinogen IX oxidase, and Ferrochetalase. These enzymes are well characterized and their role in heme biosynthesis is understood. A very important step in the production of Heme is the production of amino levulinic acid (ALA). Two anabolic pathways exist for the production of ALA, the C-4 and C-5 pathways. The enzymes of the C-4 and C-5 pathway are heme biosynthetic enzymes. An example of a C-5 pathway enzyme is glutamyl-tRNA reductase. An example of a C-4 pathway enzyme is ALA synthase.

An “ALA synthase” or “ALA sythetase” is an enzyme with the activity defined by E.C. 2.3.1.37. ALA synthase enzymes are not subject to feedback inhibition from heme. ALA synthases are a defined class of enzymes including, for example, polypeptides (SEQ ID NOs: 2 and 29-47). The identity of these SEQ IDs is shown in Table 2. The ALA synthases may be defined, for example, by shared sequence motifs as described in the Hidden Markov Model TIGR01821. An alignment of diverse ALA synthases is provided in FIG. 18. An alignment of conserved homologs of SEQ ID NO: 2 is provided in FIG. 19.

TABLE 2 Representative sample of ALA synthetases SEQ ID Species Gene product 2 Rhodobacter sphaeroides 2.4.1 HemA 29 Hyphomonas neptunium ATCC 15444 gi 114797766 30 Orientia tsutsugamushi str. Boryong gi 148284187 31 Azorhizobium caulinodans ORS 571 gi 158421958 32 Caulobacter crescentus CB15GI: 16125604 gi 16125604 33 Brucella canis ATCC 23365 gi 161618302 34 Bordetella petrii DSM 12804 gi 163855632 35 Caulobacter sp. K31 gi 167647011 36 Streptomyces griseus subsp. griseus gi 182439088 37 Orientia tsutsugamushi str. Ikeda gi 189183979 38 Phenylobacterium zucineum HLK1 gi 197105140 39 Phenylobacterium zucineum HLK1 gi 197106256 40 Caulobacter crescentus NA1000 gi 221234354 41 Candidatus Liberibacter asiaticus str. gi 254780604 42 Neorickettsia risticii str. gi 254797163 43 Brucella microti CCM 4915 gi 256368778 44 Chromobacterium violaceum ATCC 12472 gi 34496258 45 Brucella abortus bv. 1 str. gi 62289313 46 Staphylococcus aureus RF122 gi 82751601 47 Neorickettsia sennetsu str. Miyayama gi 88608338

An “ALA synthase analog” means an ALA synthase enzyme bearing one or more additions deletions or substitutions of residues compared to the original ALA synthase. Useful ALA synthase analogs include analogs which retain the activity defined by E.C. 2.3.1.37. By examining and aligning known ALA synthase sequences, a skilled person can determine which ALA synthase residues are conserved across species. Using this alignment, the skilled person could generate a consensus sequence, using, for example, the Clustal algorithm. Since conserved residues are generally those which are required for function (Boffelli D, Nobrega. M A, Rubin E M. Comparative genomics at the vertebrate extremes. Nat Rev Genet. 2004; 5:456-465), non-naturally occurring proteins that conform to this consensus sequence would define ALA synthase analogs that likely retain the ALA synthase activity defined by E.C. 2.3.1.37. For example, from the alignment in FIG. 18 or 19, a consensus sequence for several ALA synthases may be constructed. Further, additional alignments may be generated using, for example, the HomoloGene and Conserved Domain (CDD) algorithms of the National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda Md. USA.

Alternatively, generating ALA synthase analogs which retain ALA synthase activity could also be accomplished by using HMM as described above.

Interpro has one entry which describe ALA synthases: IPR010961 Tetrapyrrole biosynthesis, 5-aminolevulinic acid synthase. The J. Craig Venter Institute maintains the TIGR database. The TIGR HMM profile that describes the probable structure of ALA synthases is TIGR01821.

Although these HMMs do not provide typical sequence information regarding ALA synthase proteins, they do provide a description of the probable structure of a ALA synthase. Thus, analogs of ALA synthase that conform to the HMM would be more likely to retain ALA synthase activity. To easily generate sequences of ALA synthase analogs more likely to have ALA synthase activity, a skilled person could generate ALA synthase analog sequences using a computer to introduce substitutions, deletions or additions to a ALA synthase sequence. The relative probabilities embodied in the ALA synthase HMMs could guide a skilled person regarding which residues, when mutated, are more likely to lead to a loss of function. The skilled person could then compare the analog sequences to the HMMs in the databases listed above. Those analogs which met the threshold of being tagged as bearing a ALA synthase domain would likely have the property of ALA synthase activity. The HMMs discussed above which describe ALA synthases and ALA synthase analogs are hereby incorporated by reference.

“HemA” is the polypeptide represented by SEQ ID NO: 2.

“Growth media” or “growth medium” is a composition comprising one or more nutrients used to culture a cell. Growth medium includes any foam which accumulates in the medium.

A “vector” is a polynucleotide which can be used to introduce a desired sequence into a cell. The vector polynucleotide typically includes additional sequences, including sequences that direct propagation of the vector in the cell or insertion of part of the vector into the cell's genome, and a gene which allows an individual to screen for the presence of the vector. Common examples of vectors include plasmids, artificial chromosomes, viruses, and linear polynucleotide fragments which are designed to insert into a cell's genome. Vectors are well known tools to the skilled artisan, and an artisan can easily find appropriate vectors for a particular organism in the literature or in biobanks such as ATCC.

A “promoter” is a polynucleotide sequence that, when operably linked to a CDS, is sufficient, under one or more conditions, to cause an RNA polymerase to begin transcribing an mRNA from a polynucleotide.

A promoter is “operably linked” to a polynucleotide sequence when the promoter is linked to the polynucleotide sequence in such a way that a RNA polymerase will transcribe at least a portion of the polynucleotide sequence. Typically, linking a promoter to the 5′ end of a polynucleotide will result in the promoter being operably linked to the polynucleotide.

In the context of a first polynucleotide and a second polynucleotide, the first and second polynucleotides are “linked” when they are joined by a linker or a phosphodiester bond.

A “linker” is a polynucleotide of one of more bases which is used to link two or more polynucleotides.

“Foam” is a suspension of a gas in another substance. When foam accumulates in growth media, a gas which is in contact with the growth media become suspended in the media. For example foam often forms in vessels containing growth media when they are agitated.

Biliverdin is from a “non-animal source” when the heme oxidase catalyzed step of biliverdin production is not performed in a cell from an organism in the Eukaryotic kingdom animalia, and where the heme substrate of the heme oxidase was not produced in a cell from an organism in the Eukaryotic kingdom animalia.

“Collecting” a substance means removing a measurable quantity of the substance from the vessel in which it was produced. For example, if foam was produced in a vessel while a cell was cultured, removing the foam from the vessel is collecting the foam. Likewise, in the context of producing biliverdin, removing biliverdin from the vessel in which it was produced is collecting the biliverdin. “Collecting” does not necessitate purification of the biliverdin from the organism in which the biliverdin was synthesized.

In the context of generating a cell for producing biliverdin, “a parent cell” is a cell into which a recombinant heme oxygenase and/or a recombinant heme biosynthetic enzyme is introduced.

To “introduce” or “introducing” a polynucleotide into a cell means using physical or genetic techniques to cause the polynucleotide to enter within a membrane which surrounds the cell. These include transformation, conjugation between bacterial cells, and recombination during sexual reproduction.

To “transform” or “transforming” means to introduce a polynucleotide into a cell by chemical, electrical or other physical means. Methods of transforming cells include chemical destabilization of the cell membrane to allow the polynucleotide to enter, electroporation, microinjection, or firing particles coated with the polynucleotide into the cell.

A composition found inside a cell is “excreted” when the composition moves outside of the membrane surrounding the cell. A composition may be excreted passively, meaning that the composition diffuses across the membrane, or a compound may be excreted actively, meaning that cellular factors aid the composition in moving outside the membrane surrounding the cell.

A first composition is “located with” a second composition when a measurable quantity of the second composition is associated with the first composition such that collecting the first composition will also collect some of the associated second composition.

A polynucleotide that contains a CDS is “optimized for expression in a cell” where one or more nucleotide bases of the polynucleotide are modified according to an algorithm such that when the polynucleotide is linked to a promoter and introduced into a cell, more of the protein product of the CDS is produced than would be produced by the unmodified polynucleotide linked to the same promoter.

Various methods of modifying a polynucleotide so that it is optimized for expression in a cell are known. For example, the polynucleotide sequence may be modified according to the Codon Adaptation Index method (Sharp, P. M., and W. H. Li, (1987). The codon adaptation index a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research 15: 1281-1295, which is hereby incorporated by reference). Also, the polynucleotide sequence may be modified according to the Frequency of Optimal Codons method (Ikemura, T., (1981). Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli system. Journal of Molecular Biology 151: 389-409, which is incorporated by reference).

Alternatively, a polynucleotide may be optimized for expression in a cell where the nucleotide bases are modified according to the proprietary methods of DNA 2.0 Inc., Menlo Park, Calif.

Many of the embodiments described below incorporate methods for culturing cells, transforming cells, or performing other genetic manipulations on cells. Many methods for performing these steps are known in the art. In particular, many laboratory methods are described in Joe Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, 2001 (ISBN: 0879695773); or in Frederick M. Ausubel, et al., eds., Current Protocols in Molecular Biology, Wiley (ISSN: 1934-3639, last updated Jun. 28, 2010, which are incorporated by reference.

Methods of Producing Biliverdin

One aspect of the invention includes methods of producing biliverdin in a microorganism. Another aspect of the invention includes methods for producing biliverdin from a non-animal source. In general, both methods of producing biliverdin in a microorganism and methods of producing biliverdin from a non-animal source include culturing a cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme. The embodiments described below represent embodiments of both methods of producing biliverdin in a microorganism and methods for producing biliverdin from a non-animal source.

In one embodiment, the cultured cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase. In another embodiment, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme. The vector may be a plasmid, a construct designed to integrate into the genome of the cell, an artificial chromosome, or any other vector known in the art which is appropriate for use in the cell. For example, ATCC maintains a collection of vectors for use in a variety of organisms.

In another embodiment, a promoter may drive expression of a polynucleotide which codes for a recombinant heme oxygenase. In another embodiment, a promoter may drive expression of a polynucleotide which codes for a recombinant heme oxygenase. In any embodiment where a promoter is used, the promoter may be a regulatable promoter. Furthermore, regulatable promoters may be used to control the expression of recombinant polypeptides in a temporal or other fashion. Some regulatable promoters are inducible promoters. For example, the T7 promoter drives very low basal levels of expression when cells are grown in the absence of IPTG. However, when IPTG is added to the culture media, the promoter is activated and higher expression is induced. Other regulatable promoters may be repressible promoters. For example, the tetR promoter has very low expression when cells are grown in the presence of tetracycline, but expression increases when tetracycline is removed from the growth medium. Many promoters which are appropriate for use in a variety of cells are known in the art. The Registry of Standard Biological Parts, maintained by Massachusetts Institute of Technology discloses many promoters which will be appropriate for use in different types of cells to achieve a desired pattern of expression. The Registry of Standard Biological Parts is hereby incorporated by reference.

In another embodiment, the heme oxygenase enzyme is a HO family heme oxygenase. In another embodiment, the heme oxygenase is a HemS family heme oxygenase. In a particular embodiment, HO1 (SEQ ID NO: 1) is used as the heme oxygenase. In another embodiment, the heme oxygenase enzyme may be an HO family analog.

In another emdodiment, the heme bisynthetic enzyme used is ALA dehydratase. In another emdodiment, the heme bisynthetic enzyme used is Porphobilinogen deaminase. In another emdodiment, the heme bisynthetic enzyme used is Uroporphyrinogen III synthase. In another emdodiment, the heme bisynthetic enzyme used is Uroporphyrinogen III decarboxylase. In another emdodiment, the heme bisynthetic enzyme used is Coprophorinogen III oxidase. In another emdodiment, the heme bisynthetic enzyme used is Protopophyrinogen IX oxidase. In another emdodiment, the heme bisynthetic enzyme used is Ferrochetalase.

In certain embodiments, the heme biosynthetic enzyme used is an ALA synthase. In a particular embodiment, the heme biosynthetic enzyme is hemA (SEQ ID NO: 2). In certain other embodiments, the heme biosynthetic enzyme is an ALA synthase analog.

In a particular embodiment, the cell comprises HemA-HO1-pET101 (FIG. 9).

In another embodiment, lactose is provided to the cell which is being cultured. Generally, lactose may be provided to the cell as a component of the growth medium. Lactose may be provided in a initial concentration, and the actual concentration of lactose in the media will decrease as the cells consume it. In one embodiment, the initial concentration of lactose is from about 2% (w/v) to about 10% (w/v). In another embodiment, the lactose may be provided in an initial concentration of about 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or about 10%. In a particular embodiment the initial concentration of lactose is 2.5%.

In another embodiment, one or more trace metals is provided to the cell. The trace metal(s) may be provided in the form of a trace metal solution. In one embodiment, one or more of NaCl, ZnSO₄, MnCl₂, FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine or CaCl₂ are added to the culture. In another embodiment, NaCl, ZnSO₄, MnCl₂, FeCl₃, CuSO₄, H₃BO₃, NaMoO₄, H₂SO₄, MgSO₄, thiamine and CaCl₂ are added to the culture.

In another embodiment, the concentration of dissolved oxygen in the growth medium of the cell is measured. Any oxygen measuring device known in the art can be used. For example an oxygen measuring probe may be used. In one embodiment, InPro® 6800 Series O₂Sensors (METTLER TOLEDO) may be used in monitoring the concentration of dissolved oxygen In one embodiment, a plurality of measurements of the dissolved oxygen concentration are made. In another embodiment, the concentration of dissolved oxygen has an initial value, then the dissolved oxygen concentration decreases to a first intermediate value, then the dissolved oxygen concentration increases to a second intermediate value greater than the first intermediate value, then the dissolved oxygen concentration decreases to a third intermediate value less than the first intermediate value.

In another embodiment, the biliverdin is excreted from the cell into the growth medium. In another embodiment, foam accumulates in the growth medium, and a portion of the biliverdin is located with the foam. In a related embodiment, the foam is collected. In another embodiment, the biliverdin is collected.

For any embodiment of a method of producing biliverdin discussed above, the cell may comprise a polynucleotide that is optimized for expression in a cell

In certain embodiments, the cell comprises a sequence that codes for a heme oxygenase that is optimized for expression in a cell. In certain embodiments, the cell comprises a sequence that codes for an HO family heme oxygenase that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in a bacterial cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in an Escherichia coli cell. In another embodiment, the cell comprises a polynucleotide comprising SEQ ID NO: 50, wherein the cell is a bacterial cell. In another embodiment, the cell comprises a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is a bacterial cell. In another embodiment, the cell comprises a polynucleotide comprising SEQ ID NO: 50, wherein the cell is an Escherichia coli cell. In another embodiment, the cell comprises a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is an Escherichia coli cell.

In certain embodiments, the cell comprises a sequence that codes for a heme biosynthetic enzyme that is optimized for expression in a cell. In certain embodiments the cell comprises a sequence that codes for an ALA synthase that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in a cell. In a related embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in a bacterial cell. In a related embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in an Escherichia coli cell.

Cells for Producing Biliverdin

Another aspect of the invention includes cells for producing biliverdin. In general, cells for producing biliverdin comprise a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.

In one embodiment, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase. In another embodiment, the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme. The vector may be a plasmid, a construct designed to integrate into the genome of the cell, an artificial chromosome, or any other vector known in the art which is appropriate for use in the cell. For example, ATCC maintains a collection of vectors for use in a variety of organisms.

In another embodiment, the cell comprises a promoter that may drive expression of a polynucleotide which codes for a recombinant heme oxygenase. In another embodiment, the cell comprises a promoter that may drive expression of a polynucleotide which codes for a recombinant heme oxygenase. In any embodiment where a promoter is used, the promoter may be a regulatable promoter. Furthermore, regulatable promoters may be used to control the expression of recombinant polypeptides in a temporal or other fashion. Some regulatable promoters are inducible promoters. For example, the T7 promoter drives very low basal levels of expression when cells are grown in the absence of IPTG (FIG. 8). However, when IPTG is added to the culture media, the promoter is activated and higher expression is induced. Other regulatable promoters may be repressible promoters. For example, the tetR promoter has very low expression when cells are grown in the presence of tetracycline, but expression increases when tetracycline is removed from the growth medium. Many promoters which are appropriate for use in a variety of cells are known in the art. The Registry of Standard Biological Parts, maintained by Massachusetts Institute of Technology discloses many promoters which will be appropriate for use in different types of cells to achieve a desired pattern of expression. The Registry of Standard Biological Parts is hereby incorporated by reference.

In another embodiment, the heme oxygenase enzyme is a HO family heme oxygenase. In another embodiment, the heme oxygenase is a HemS family heme oxygenase. In a particular embodiment, HO1 (SEQ ID NO: 1) is used as the heme oxygenase. In another embodiment, the heme oxygenase enzyme may be an HO family analog.

In another emdodiment, the heme bisynthetic enzyme used is ALA dehydratase. In another emdodiment, the heme bisynthetic enzyme used is Porphobilinogen deaminase. In another emdodiment, the heme bisynthetic enzyme used is Uroporphyrinogen III synthase. In another emdodiment, the heme bisynthetic enzyme used is Uroporphyrinogen III decarboxylase. In another emdodiment, the heme bisynthetic enzyme used is Coprophorinogen III oxidase. In another emdodiment, the heme bisynthetic enzyme used is Protopophyrinogen IX oxidase. In another emdodiment, the heme bisynthetic enzyme used is Ferrochetalase.

In certain embodiments, the heme biosynthetic enzyme used is an ALA synthase. In a particular embodiment, the heme biosynthetic enzyme is hemA (SEQ ID NO: 2). In certain other embodiments, the heme biosynthetic enzyme is an ALA synthase analog.

In a particular embodiment, the cell comprises HemA-HO1-pET101 (FIG. 9).

For any embodiment that provides a cell for producing biliverdin discussed above, the cell may comprise a polynucleotide that is optimized for expression in a cell.

In certain embodiments, the cell comprises a sequence that codes for a heme oxygenase that is optimized for expression in a cell. In certain embodiments, the cell comprises a sequence that codes for an HO family heme oxygenase that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in a bacterial cell. In one embodiment, the cell comprises a sequence that codes for HO-1 that is optimized for expression in an Escherichia coli cell. In another embodiment, the cell comprises a polynucleotide comprising SEQ ID NO: 50, wherein the cell is a bacterial cell. In another embodiment, the cell comprises a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is a bacterial cell. In another embodiment, the cell comprises a polynucleotide comprising SEQ ID NO: 50, wherein the cell is an Escherichia coli cell. In another embodiment, the cell comprises a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50, wherein the cell is an Escherichia coli cell.

In certain embodiments, the cell comprises a sequence that codes for a heme biosynthetic enzyme that is optimized for expression in a cell. In certain embodiments the cell comprises a sequence that codes for an ALA synthase that is optimized for expression in a cell. In one embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in a cell. In a related embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in a bacterial cell. In a related embodiment, the cell comprises a sequence that codes for HemA that is optimized for expression in an Escherichia coli cell.

Methods of Producing Cells

Another aspect of the invention includes methods of producing cells for producing biliverdin. In general, these methods comprise introducing into a cell a recombinant polypeptide. In one embodiment, a recombinant heme oxygenase is introduced into the cell. In another embodiment, a recombinant heme biosynthetic enzyme is introduced into the cell.

In one embodiment, the method comprises introducing into the cell a polynucleotide comprising a sequence which codes for a recombinant heme oxygenase. In another embodiment, the method comprises introducing into the cell a polynucleotide comprising a sequence which codes for a recombinant heme biosynthetic enzyme. In another embodiment, a polynucleotide is introduced into the cell, the polynucleotide comprising a first sequence which codes for a recombinant heme oxygenase and a second sequence which codes for a recombinant heme biosynthetic enzyme. In a particular embodiment, the HemA-HO1-pET101 (FIG. 9) is introduced into the cell.

In another embodiment, a promoter is introduced into the cell in such a way that it recombines with a sequence which codes for a heme oxygenase to produce a linked polynucleotide which codes for a recombinant heme oxygenase, where the parent cell comprises a recombinant heme biosynthetic enzyme. In another embodiment, a promoter is introduced into the cell in such a way that it recombines with a sequence which codes for a heme biosynthetic enzyme to produce a linked polynucleotide which codes for a recombinant heme biosynthetic enzyme, where the parent cell comprises a recombinant heme oxygenase. Methods of introducing polynucleotides for directed recombination with polynucleotides in a cell are known in the art. For example, methods for directed recombination are discussed in AJ Link et al., Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization, J. Bacteriol, (179) 6228-6237 (1997), which is incorporated by reference.

In another embodiment, any step comprising introducing a polynucleotide into a cell comprises transforming the cell.

In another embodiment, the cell is a microorganism. In another embodiment, the cell is a prokaryotic cell. In another embodiment, the cell is a bacterial cell. In another embodiment, the cell is an Escherichia coli cell.

For any embodiment that provides a method of producing a cell for producing biliverdin discussed above, the polynucleotides may be optimized for expression in a cell.

In certain embodiments, a sequence that codes for a heme oxygenase that is optimized for expression in a cell is introduced into a cell. In certain embodiments, a sequence that codes for an HO family heme oxygenase that is optimized for expression in a cell is introduced into a cell. In one embodiment, a sequence that codes for HO-1 that is optimized for expression in a cell is introduced into a cell. In another embodiment, a polynucleotide comprising SEQ ID NO: 50 is introduced into a bacterial cell. In another embodiment, a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50 is introduced into a bacterial cell. In another embodiment, a polynucleotide comprising SEQ ID NO: 50 is introduced into an Escherichia coli cell. In another embodiment, a polynucleotide comprising bases 15 to 776 of SEQ ID NO: 50 is introduced into an Escherichia coli cell.

In certain embodiments, a sequence that codes for a heme biosynthetic enzyme that is optimized for expression in a cell is introduced into a cell. In certain embodiments a sequence that codes for an ALA synthase that is optimized for expression in a cell is introduced into a cell. In one embodiment, a sequence that codes for HemA that is optimized for expression in a cell is introduced into a cell. In a related embodiment, a sequence that codes for HemA that is optimized for expression in a cell is introduced into a bacterial cell. In a related embodiment, a sequence that codes for HemA that is optimized for expression in a cell is introduced into an Escherichia coli cell.

The following examples are intended to further illustrate exemplary embodiments and are not intended to limit the scope of the disclosure.

EXAMPLES Example 1 Construction of Plasmid Expression Vectors and Gene Expression

HO-1 pET101: HO(HO-1) gene of Synechocystis PCC6803 was amplified by the polymerase chain reaction (PCR) using the following primers:

(HO1 forward primer) CACCATGAGTGTCAACTTAGCTTC (SEQ ID NO: 3) (HO1 reverse primer) CTAGCCTTCGGAGGTGGCGA (SEQ ID NO: 4)

The PCR product was blunt ended using thermostable proofreading polymerase, gel purified, ligated into pET101 vector by directional TOPO® Cloning Reaction and transformed into chemically competent E. coli TOP10 (Invitrogen) cells according to the manufacturer's instructions. Five white colonies were selected on Xgal agar plates, plasmids isolated and subjected to gel electrophoresis to confirm cloning of HO-1 into the vector. DNA sequencing showed that the cloned DNA had an identical sequence to Synechococcus PCC6803 HO-1 (SEQ ID NO: 48) (FIG. 6). The vector with the HO-1 gene was transformed into E. coli BL21 (Invitrogen) and cells from a single white colony were propagated in Luria-Bertani (LB) broth medium (25 g per L, Fisher Scientific) plus 100 μg per mL ampicillin. Its DNA plasmids were isolated and the HO-1 gene sequence in the plasmid was confirmed by DNA sequencing again. The plasmid was used as the clone that harbored expression vector HO-1 pET101.

HemA-HO-1 pET101: HemA which encodes ALA synthase from R. sphaeroides was amplified by the polymerase chain reaction (PCR) using the following primers:

(Hem A forward primer) (SEQ ID NO: 5) ACAACGTTGAAGGAGCCCTTCTCCATGGACTACAATCTGGCACT (Hem A reverse primer) (SEQ ID NO: 6) ATGACCGGTACGTCAGGCAACGACCTCGGCGC

The HemA gene was cut by restriction enzymes (AclI and AgeI) and ligated to HO1-pET101 vector which was digested by restriction enzymes (BstBI and AgeI). The construct was transformed into competent E. coli BL21 (DE3) (Invitrogen) cells according to the manufacturer's instructions. Five white colony isolates were selected and were propagated in Luria-Bertani (LB) broth medium (25 g per L, Fisher Scientific) plus 100 μg per mL ampicillin. Their plasmid DNAs were extracted and the occurrence of HemA was confirmed by DNA sequencing (FIG. 7—SEQ ID NO: 49). After DNA sequencing analysis of the plasmid DNAs, an isolate yielding plasmid DNA with the expected size was selected, designated E. coli strain HemA-HO-1 and was used as the clone that harbors expression vector HemA-HO-1 pET101.

Example 2 Bacterial Growth, Protein Expression, and Production of Biliverdin

E. coli strains HO-1 and HemA-HO-1 were maintained on LB agar medium with 100 μg per mL ampicillin. For analysis of protein expression, cells were grown in LB broth medium supplemented with 1% glucose and 100 μg per mL ampicillin with rotary shaking 225 rpm at 37° C. overnight. IPTG was added (1 mM final concentration) when the culture achieved an absorbance between 0.3 and 0.5 at 600 nm. Exponentially grown cells were harvested and lysed and cell extracts were recovered as supernatant fractions after centrifugation at 8,000×g for a minimum of 5 min. The cell extracts were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis and the gel was stained with Coomasie Brilliant Blue. The stained gel showed the induction of the HO-1 protein (23 kdaltons) by IPTG in both E. coli strains which confirmed the expression of HO-1 gene.

For biliverdin production, the HemA and HO-1 containing E. coli strains are grown in 100 to 200-ml of LB broth medium plus 100 μg per mL ampicillin in 500-mL capacity Erlenmeyer flasks in a rotary water bath shaker (200 rpm) at 37° C. to a cell density showing an absorbance of 4 to 5 measured at 600 nm using a 1 cm path length cuvette and LB broth medium as blank. The culture is then added to 1 or 1.5 L of LB broth medium or biliverdin—Minimal Medium (biliverdin 2) (per L, KH₂PO₄, 3.5 g; K₂HPO₄, 5.0 g; (NH₄)₂SO₄, 5.0 g; yeast extract, 5.0 g; trace metals solution (per L, NaCl, 5 g; ZnSO₄-7H₂O, 1 g; MnCl₂-4H₂O, 4 g; FeCl₃-6H₂O, 4.75 g; CuSO₄-5H₂O, 0.4 g; H₃BO₃, 0.575 g; NaMoO₄-2H₂O, 0.5 g; and 6N H₂SO₄, ˜12.5 ml), 1 mL; MgSO₄-7H₂O (25%), 4 mL; thiamine (220 mg per mL), 10 mL, CaCl₂-H₂O (15 g per L), 10 mL with trace metals solution, MgSO₄-7H₂O, thiamine, and CaCl₂-H₂O filter sterilized separately and added to the other ingredients after autoclaving) containing 100 mg per L ampicillin and 2.5% α-lactose both filtered sterilized and added separately after autoclaving. The final pH for both growth media is 7.0. The E. coli inocula cultures are added to give an initial absorbance of ˜0.03 measured at 600 nm using a 1 cm path length cuvette. The cultures are grown in a VirTis Omniculture bioreactor system with 2.0 L vessel (VirTis, Gardner, N.Y.). Airflow is at 3.5 liters per min at 30° C. Agitation is set at 250 rpm until an absorbance of 0.4 to 0.5 measured at 600 nm (1 cm path length) is attained, and then agitation is increased to 450 rpm. Under these conditions, blue-green pigmented material becomes visible 6 to 10 hours at or near the top of foam formed above the surface of the culture. At this point, dissolved oxygen levels are less than 5% saturation. The material accumulates as a blue-green film and as blue-green aggregates that adhere to the glass walls of the vessel and the stainless steel head plate. In addition, polyethylene tubing (Tygon, ¼ inch I.D., 0.5 to 1 m length) connected to an outlet port of the vessel head plate and with the other end opened into a receiving flask is used to collect foam-trapped blue-green material during culture growth. Growth is terminated after approximately 20 hours or when production of blue-green material ceases.

Example 3 Collecting the Biliverdin

After the growth of a batch culture is completed, the blue-green film and aggregated materials are physically removed from the vessel surfaces using a spatula and suspended in methanol. The methanol suspension is combined with the blue green material collected in the receiving flask and an equivalent volume of methanol is added. A volume of 1N HCl equal to the total volume of the methanol suspension is added dropwise to the suspension with stirring. A 1/10 volume of water is added, and the mixture is vortexed and extracted into chloroform. The green chloroform layer is recovered, dried, and the resulting blue-green material is stored in the dark at −20° C. Absorbance spectra of this material resemble the spectrum obtained for authentic biliverdin (Frontier Scientific, Inc.) suggesting that the blue-green material contains biliverdin IXα. For purification, the dried material is dissolved in 40% methanol, 0.2 M Na acetate, pH 5.2 and loaded onto a Sepak C18 column. The column is eluted successively with 40% methanol, 0.2 M Na acetate, pH 5.2, water, and 100% methanol. The blue-green material elutes with 100% methanol (FIG. 10) and is collected. It is acidified with HCl (0.25 N final concentration), extracted into chloroform and stored in the dark at −20° C.

Example 4 Analytical Determination of Biliverdin

Absorbance spectra between wavelengths 300 and 800 nm were obtained using an Applied Biosystem spectrophotometer. The presence of biliverdin in the blue-green material is evident by comparison to the absorbance spectrum of a known standard of biliverdin (from Frontier Scientific, Inc.) derived from bilirubin IXα (FIG. 11) with characteristic spectral peaks at 385 nm and 650 nm-660 nm. The same absorbance spectrum was obtained with blue-green material produced by E. coli strains TOP10 or BL21 transformed with and expressing HO1-pET101 or HemA-HO-1 pET101, respectively. Also, the same absorbance spectrum was obtained with blue-green material from cultures of E. coli strains grown in either LB broth medium or biliverdin 2 broth medium. High performance liquid chromatography (HPLC) is also an analytical tool to help determine the chemical identity of the purified blue-green material as biliverdin. Identical or very similar HPLC peaks (major peaks with Rf values of 13.19 and 13.17 in FIG. 12) monitored at 385 nm are obtained with the blue-green material produced by E. coli strains TOP10 or BL21 transformed with and expressing HO1-pET101 or HemA-HO-1 pET101, respectively (FIG. 12). An HPLC peak with an Rf value of 13.19 is obtained with the biliverdin commercial standard indicating that the blue-green material contains biliverdin in high concentration. Finally, mass spectral analyses of the purified blue-green material reveal a predominant molecular mass of 583.2-583.3 which is the expected monoisotopic mass of biliverdin IXα and that was also observed with the biliverdin IXα commercial standard (FIG. 13). Altogether, the analytical results show that E. coli strains TOP10 or BL21 transformed with and expressing HO1-pET101 or HemA-HO-1 pET101, respectively, produce biliverdin IXα when grown as described.

Example 5 Other Factors which Influence the Production of Biliverdin

The effect of trace metals on biliverdin production was tested. A trace metal solution consisting of (per L) NaCl, 5 g; ZnSO₄-7H₂O, 1 g; MnCl₂-4H₂O, 4 g; FeCl₃-6H₂O, 4.75 g; CuSO₄-5H₂O, 0.4 g; H₃BO₃, 0.575 g; NaMoO₄-2H₂O, 0.5 g; and 6N H₂SO₄, ˜12.5 ml) was added to the growth medium. The amount of biliverdin recovered with different amounts of trace metal solution added is reported in Table 3.

TABLE 3 Effect of trace metal solution addition on biliverdin production trace metals solution Average amount BVIXα Range BVIXα (mL L⁻¹) produced (mg L⁻¹) produced (mg L⁻¹) 0 <0.1 <0.1 1.0 7.0 2.0-9.2 2.0 12 4.5-20

The concentration of dissolved oxygen during biliverdin production was also tested. dO₂ was measured by InPro® 6800 Series O₂Sensors (METTLER TOLEDO) connected to BIOFLO® 310 Fermentation System (New Brunswick Scientific). The results are plotted in FIG. 15. In general, the dissolved oxygen profile seen in FIG. 15 is associated with the production of biliverdin. In particular, the small spike (or peak) in oxygen concentration seen at about 5:07:40 is correlated with biliverdin production. Thus, if the dissolved oxygen concentration decreases from its initial value to a first intermediate value, then increases to a second intermediate value higher than the first intermediate value, then decreases to a third intermediate value lower than the first intermediate value, it is expected that the cells in the culture will produce biliverdin.

Example 6 Biliverdin Production Using an Optimized Polynucleotide Construct

The gene coding for HO1 was optimized to work in E. coli and synthesized by DNA 2.0 Inc. (Menlo Park, Calif.). The coding sequence for a 6×His tag was incorporated at the 5′ end of the sequence so as to introduce six histidines to the N-terminus of HO1 expressed from the construct (FIG. 20). The synthesized HO1 gene was inserted into Vector pJexpress 401 (DNA 2.0, Menlo Park, Calif.) to form vector HO1-pJ401 (FIG. 22). The vector HO1-pJ401 was transformed into E. coli BL21.

For biliverdin production, modified HO1-pJ401 containing E. coli strains were grown in 80-ml of LB broth medium plus 100 μg per mL Kanamycin in 250-mL capacity Erlenmeyer flasks in a shaker (225 rpm) at 37° C. to a cell density showing an absorbance of 2 to 6 measured at 600 nm using a 1 cm path length cuvette and LB broth medium as blank. The culture was then added to 2 L medium with 20 g NZamineA (Amersco, Solon Ohio), 10 g Yeast extract (Fisher), 10 ml 100% Glycerol (Amersco, Solon Ohio), 20 ml Lactose 20%, 5 ml Glucose 20%, 10 ml 200× Trace elements, 1 ml 2M MgSO₄, and 100 ml 20× NPS (Studier 2006) in NewBrunswick Bioflow 310 controller used with bioCamand software for data collection, 40% dissolved oxygen cascade control (0-30 percent O₂ supplemented to airflow, 280-500 rpm agitation, and 0.75-4-slpm airflow), and exponential feed, 200 ml 10% glycerol and 2% lactose feed. Blue-green pigmented material became visible 4 to 10 hours after lactose induction at or near the top of the foam formed above the surface of the culture. Production of biliverdin increased about two fold or more compared to the production in Example 2.

The 200× trace elements solution was prepared by adding to a final volume of 250 ml: 0.5 ml HCl in 50 ml H₂O, FeCl₃ 0.675 g, CaCl₂ 0.15 g, MnCl₂ 0.1 g, ZnSO₄ 0.015 g, CoCl₂ 0.023 g, CuCl₂ 0.015 g, NiCl₂ 0.023 g, Na₂MoO₄ 0.025 g, and H₃BO₃ 0.007 g, then filter sterilizing. The 20× NPS stock solution was prepared by dissolving 66 g (NH₄)₂SO₄, 136 g KH₂PO₄, and 142 g Na₂HPO₄ in 1 L dd H₂O. The solution was autoclaved at 121° C. 15 min.

It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims. 

1. A method of producing biliverdin from a non-animal source, comprising: culturing a cell in a growth medium, the cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.
 2. The method of claim 1, wherein the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme oxygenase.
 3. The method of claim 1, wherein the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme.
 4. The method of claim 1, wherein the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme oxygenase.
 5. The method of claim 1, wherein the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme biosynthetic enzyme.
 6. The method of claim 2, wherein the polynucleotide which codes for the heme oxygenase comprises SEQ ID NO:
 50. 7. The method of claim 1, wherein the heme biosynthetic enzyme is an ALA synthase.
 8. The method of claim 1, wherein the heme oxygenase is a HO family enzyme.
 9. The method of claim 1, wherein the heme biosynthetic enzyme is hemA.
 10. The method of claim 1, wherein the heme oxygenase enzyme is HO1.
 11. The method of claim 1, further comprising the step of taking at least an initial measurement, a first intermediate measurement, a second intermediate measurement, and a third intermediate measurement of dissolved oxygen concentration in the growth medium of the cell, wherein the initial measurement yields an initial value, the first intermediate measurement yields a first intermediate value less than the initial value, the second intermediate measurement yields a second intermediate value greater than the first intermediate value, and the third intermediate measurement yields a third intermediate value less than the first intermediate value, and wherein dissolved oxygen in the growth medium in concentrations at the initial value, the first intermediate value, the second intermediate value and the third intermediate value indicates that biliverdin will be produced.
 12. The method of claim 1, further comprising the step of collecting the biliverdin.
 13. A cell comprising a recombinant heme oxygenase and a recombinant heme biosynthetic enzyme.
 14. The cell of claim 13, wherein the cell comprises a vector comprising a polynucleotide which codes for the recombinant heme oxygenase.
 15. The cell of claim 14, wherein the polynucleotide which codes for the recombinant heme oxygenase comprises SEQ ID NO:50.
 16. The cell of claim 13, wherein the cell comprises a vector comprising a polynucleotide which codes for a recombinant heme biosynthetic enzyme.
 17. The cell of claim 13, wherein the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme oxygenase.
 18. The cell of claim 13, wherein the cell comprises a regulatable promoter operably linked to a polynucleotide which codes for the heme biosynthetic enzyme.
 19. The cell of claim 13, wherein the heme biosynthetic enzyme is an ALA synthase.
 20. The cell of claim 13, wherein the heme oxygenase is a HO family enzyme.
 21. The cell of claim 13, wherein the heme biosynthetic enzyme is hemA.
 22. The cell of claim 13, wherein the heme oxygenase enzyme is HO1.
 23. The cell of claim 13, wherein the cell is an Escherichia coli cell.
 24. A method of producing the cell of claim 13, comprising: introducing into a parent cell a polynucleotide comprising a sequence which codes for a recombinant heme oxygenase, wherein the parent cell comprises a recombinant heme biosynthetic enzyme.
 25. The method of claim 24, wherein the polynucleotide comprising a sequence which codes for a recombinant heme oxygenase comprises SEQ ID NO:
 50. 26. A method of producing the cell of claim 13, comprising: introducing into a parent cell a polynucleotide comprising a sequence which codes for a recombinant heme biosynthetic enzyme, wherein the parent cell comprises a recombinant heme oxygenase.
 27. A method of producing the cell of claim 13, comprising: introducing into a parent cell a polynucleotide comprising i. a first sequence which codes for a recombinant heme oxygenase; and ii. a second sequence which codes for a recombinant heme biosynthetic enzyme. 